The present invention relates to the field of paramyxoviridae vaccines and is particularly concerned with vaccines comprising RNA encoding the fusion (F) protein of respiratory syncytial virus (RSV).
Human respiratory syncytial virus (RSV) has been identified as a major pathogen responsible for severe respiratory tract infections in infants, young children and the institutionalized elderly (refs. 1,2,3,4xe2x80x94throughout this application, various references are cited in parentheses to describe more fully the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). Global mortality and morbidity figures indicate that there is an urgent need for an efficacious RSV vaccine (refs. 5,6). In the USA alone, approximately 100,000 children are hospitalized annually with severe cases of pneumonia and bronchiolitis resulting from an RSV infection. Inpatient and ambulatory care for children with RSV infections has been estimated to cost in excess of $340 million each year in the USA. The World Health Organization (WHO) and the National Institute of Allergy and Infectious Disease (NIAID) vaccine advisory committees have ranked RSV second only to HIV for vaccine development. Both the annual morbidity and mortality figures as well as the staggering health care costs for managing RSV infections have provided the incentive for aggressively pursuing the development of efficacious RSV vaccines. However, such a vaccine is still not available.
Formalin-inactivated (FI-RSV) and live attenuated RSV vaccines have failed to demonstrate efficacy in clinical trials (refs. 7,8,9,10). Moreover, the formalin-inactivated RSV vaccine caused enhanced disease in some children following exposure to wild-type RSV (refs. 7,8,9,10). Elucidation of the mechanism(s) involved in the potentiation of RSV disease is important for the design of safe RSV vaccines, especially for the seronegative population. Recent experimental evidence suggests that an imbalance in cell-mediated responses may contribute to immunopotentiation. Enhanced histopathology observed in mice that were immunized with the FI-RSV and challenged with virus could be abrogated by depletion of CD4+ cells or both interleukin-4 (IL-4) and IL-10.
The RSV fusion (F) glycoprotein is one of the major immunogenic proteins of the virus. This envelope glycoprotein mediates both fusion of the virus to the host cell membrane and cell-to-cell spread of the virus (ref. 1). The F protein is synthesized as a precursor (F0) molecule which is proteolytically cleaved to form a disulphide-linked dimer composed of the N-terminal F2 and C-terminal F1 moieties (ref. 11). The amino acid sequence of the F protein is highly conserved among RSV subgroups A and B and is a cross-protective antigen (refs. 6,12). In the baculovirus expression system, a truncated secreted version of the RSV F protein has been expressed in Trichoplusia ni insect cells (ref. 13). The recombinant protein was demonstrated to be protective in the cotton rats (ref. 13).
Studies on the development of live viral vaccines and glycoprotein subunit vaccines against parainfluenza virus infection are being pursued. Clinical trial results with a formalin-inactivated PIV types 1,2,3 vaccine demonstrated that this vaccine was not efficacious (refs. 14, 15, 16). Further development of chemically-inactivated vaccines was discontinued after clinical trials with a formalin-inactivated RSV vaccine demonstrated that not only was the vaccine not effective in preventing RSV infection but many of the vaccinees who later became infected with RSV suffered a more serious disease. Most of parainfluenza vaccine research has focussed on candidate PIV-3 vaccines (ref. 17) with significantly less work being reported for PIV-1 and PIV-2. Recent approaches to PIV-3 vaccines have included the use of the closely related bovine parainfluenza virus type 3 and the generation of attenuated viruses by cold-adaptation of the virus (refs. 18, 19, 20, 21).
Another approach to parainfluenza virus type 3 vaccine development is a subunit approach focusing on the surface glycoproteins hemagglutinin-neuraminidase (HN) and the fusion (F) protein (refs. 22, 23, 24). The HN antigen, a typical type II glycoprotein, exhibits both haemagglutination and neuraminidase activities and is responsible for the attachment of the virus to sialic acid containing host cell receptors. The type I F glycoprotein mediates fusion of the viral envelope with the cell membrane as well as cell to cell spread of the virus. It has recently been demonstrated that both the HN and F glycoproteins are required for membrane fusion. The F glycoprotein is synthesized as an inactive precursor (F) which is proteolytically cleaved into disulfide-linked F2 and F1 moieties. While the HN and F proteins of PIV-1, -2 and -3 are structurally similar, they are antigenically distinct. Neutralizing antibodies against the HN and F proteins of one of PIV type are not cross-protective. Thus, an effective PIV subunit vaccine must contain the HN and F glycoproteins from the three different types of parainfluenza viruses. Antibody to either glycoprotein is neutralizing in vitro. A direct correlation has been observed between the level of neutralizing antibody titres and resistance to PIV-3 infections in infants. Native subunit vaccines for parainfluenza virus type 3 have investigated the protectiveness of the two surface glycoproteins. Typically, the glycoproteins are extracted from virus using non-ionic detergents and further purified using lectin affinity or immunoaffinity chromatographic methods. However, neither of these techniques may be entirely suitable for large scale production of vaccines under all circumstances. In small animal protection models (hamsters and cotton rats), immunization with the glycoproteins was demonstrated to prevent infection with live PIV-3 (refs. 25, 26, 27, 28, 29). The HN and F glycoproteins of PIV-3 have also been produced using recombinant DNA technology. HN and F glycoproteins have been produced in insect cells using the baculovirus expression system and by use of vaccinia virus and adenovirus recombinants (refs. 30, 31, 32, 33, 34). In the baculovirus expression system, both full-length and truncated forms of the PIV-3 glycoproteins as well as a chimeric F-HN fusion protein have been expressed. The in recombinant proteins have been demonstrated to be protective in small animal models (see W091/00104, U.S. application Ser. No. 07/773,949 filed Nov. 29, 1991, assigned to the assignee hereof).
Semliki Forest virus (SFV) is a member of the Alphavirus genus in the Togaviridae family. The mature virus particle contains a single copy of a ssRNA genome with a positive polarity that is 5xe2x80x2-capped and 3xe2x80x2-polyadenylated. It functions as an mRNA and naked RNA can start an infection when introduced into cells. Upon infection/transfection, the 5xe2x80x2 two-thirds of the genome is translated into a polyprotein that is processed into the four nonstructural proteins (nsP1 to 4) by self cleavage. Once the ns proteins have been synthesized they are responsible for replicating the plus-strand (42S) genome into full-length minus strands (ref. 35). These minus-strands then serve as templates for the synthesis of new plus-strand (42S) genomes and the 26S subgenomic mRNA (ref. 35). This subgenomic mRNA, which is colinear with the last one-third of the genome, encodes the SFV structural proteins. In 1991 Liljestrom and Garoff (ref. 36) designed a series of expression vectors based on the SFV cDNA replicon. These alphavirus vectors also are described in WO 92/10578, the disclosure of which is incorporated herein by reference. These vectors had the virus structural protein genes deleted to make the way for heterologous inserts, but preserved the nonstructural coding region for production of the nsP1 to 4 replicase complex. Short 5xe2x80x2 and 3xe2x80x2 sequence elements required for RNA replication were also preserved. A polylinker site was inserted downstream from the 26S promoter followed by translation stop sites in all three frames. An SpeI site was inserted just after the 3xe2x80x2 end of the SFV cDNA for linearization of the plasmid for use in vitro transcription reactions.
Injections of SFV RNA encoding a heterologous protein have been shown to result in the expression of the foreign protein and the induction of antibody in a number of studies (refs. 37, 38). The use of SFV RNA inoculation to express foreign proteins for the purpose of immunization would have several of the advantages associated with plasmid DNA immunization. For example, SFV RNA encoding a viral antigen may be introduced in the presence of antibody to that virus without a loss in potency due to neutralization by antibodies to the virus. Also, because the protein is expressed in vivo the protein should have the same conformation as the protein expressed by the virus itself. Therefore, concerns about conformational changes which could occur during protein purification leading to a loss in immunogenecity, protective epitopes and possibly immunopotentiation, could be avoided by nucleic acid immunization.
Immunization with SFV RNA also has several unique advantages over plasmid DNA immunization. SFV is one of the most efficiently replicating viruses known. After a few hours, up to 200,000 copies of the plus-RNAs can be made in a single cell. These SFV RNAs are so abundant almost all of the cells ribosomes are enrolled in the synthesis of the SFV encoded proteins, thus overtaking host cell protein synthesis (ref. 36). Therefore, it should require a smaller dose of SFV RNA and less time to achieve a protective effect as compared to plasmid DNA immunization. Secondly, RNA, unlike DNA, poses no potential threat of integrating into the cell genome. Thirdly, SFV RNA replication and expression occurs only in the cytoplasm of the cell. Therefore, problems involving nuclear transport and splicing associated with nucleus-based expression systems (DNA immunization) are absent. Fourthly, since the replication of the SFV RNA is transient and RNA is quite labile, the SFV RNA will not persist for long periods after immunization like DNA plasmids.
In WO 95/27044, the disclosure of which is incorporated herein by reference, there is described the use of alphavirus cDNA vectors based on cDNA complementary to the alphavirus RNA sequence. Once transcribed from the cDNA under transcriptional control of a heterlogous promoter, the alphavirus RNA is able to self-replicate by means of its own replicase and thereby amplify the copy number of the transcribed recombinant RNA molecules.
In copending U.S. patent application Ser. No. 08/476,397 filed Jun. 7, 1995, now U. S. Pat. No. 6,019,980 (WO 96/40945), assigned to the assignee hereof and the disclosure of which is incorporated herein by reference, there are described certain plasmid constructs used for DNA immunization which include forms of the RSV F gene. As seen therein, one plasmid pXL2 conferred complete protection on mice to challenge by live RSV when administered intranasally. This plasmid contains a gene encoding a truncated RSV F protein lacking the transmembrane portion of the protein, the immediate-early promoter enhancer and intron sequences of human cytomegatrovius (CMV) and the intron II sequences of rabbit xcex2-globin to prevent aberrant splicing. The same plasmid construct but without the intron II sequences of rabbit xcex2-globin, i.e. pXL1, provided only partial protection. Similarly, plasmid construct pXL4, which is the same as pXL2 except the RSV F gene encodes the full length RSV protein, provided partial protection while the corresponding construct lacking the intron II sequence of rabbit xcex2-globin, i.e. pXL3, conferred no protection.
These data show that the absence of elements to reduce aberrant splicing adversely affects the, protective ability of the plasmid. Aberrant splicing occurs during nuclear transcription of DNA to RNA. By employing RNA transcripts for immunization, the need for nuclear processing is avoided and aberrant splicing is unable to occur. This enables the use of the intron II sequences from non-human sources to be avoided.
The use of RNA transcripts for administration to the host enables there to be obtained total protection to challenge using a lower dose in less time than when employing the DNA plasmids described in U.S. Ser. No. 08/476,397 (WO 96/40945). The use of the RNA transcripts avoids persistence of DNA in the immunized host and potential integration.
The ability to immunize against disease caused by RSV by immunization with naked SFV RNA encoding the RSV F protein, particularly the secreted version of the RSV F protein, was unknown before the present invention and could not be predicted on the basis of the known prior art. Infection with RSV leads to serious disease. It would be useful and desirable to provide improved vectors for in vivo administration of immunogenic preparations, including vaccines, for protection against disease caused by RSV. In particular, it would be desirable to provide vaccines that are immunogenic and protective in the elderly and paediatric human populations, including seronegative infants, that do not cause disease enhancement (immunopotentiation).
The present invention provides novel immunogenic materials and immunization procedures based on such novel materials for immunizing against disease caused by paramyxoviridae, including respiratory syncytial virus and parainfluenza virus. In particular, the present invention is directed towards the provision of RNA vaccines against disease caused by infection with paramyxoviridae.
In accordance with one aspect of the present invention, there is provided a vector, comprising a first DNA sequence which is complementary to at least part of an alphavirus RNA genome and having the complement of complete alphavirus RNA genome replication regions; a second DNA sequence encoding a paramyxovirus protein or a protein fragment that generates antibodies that specifically react with the paramyxovirus protein; the second DNA sequence being inserted into a region of the first DNA sequence which is non-essential for replication; the first and second DNA sequences being under transcriptional control of a promoter.
The paramyxovirus protein may be selected from the group consisting of a parainfluenza virus (PIV) and a respiratory syncytial virus (RSV). The PIV protein may be PIV-1, PIV-2, PIV-3 or PIV-4, particularly the HN or F glycoproteins of PIV-3. The RSV protein particularly may be the F or G glycoprotein of RSV.
The second DNA sequence may encode a full length RSV F protein, or may encode a RSV F protein lacking the transmembrane anchor and cytoplasmic tail. The lack of the coding region for the transmembrane anchor and cytoplasmic tail results in a secreted form of the RSV F protein.
The second DNA sequence preferably encodes a RSV F protein and lacks a SpeI restriction site, and optionally, also lacking the transmembrane anchor and cytoplasmic tail encoding region. The absence of the SpeI restriction site may be carried out by mutating nucleotide 194 (T) of the RSV F gene to a C, which eliminates the SpeI without altering the amino acid sequence. The nucleotide sequence (SEQ ID No: 1) and encoded amino acid sequence (SEQ ID No: 2) of the mutated truncated RSV F gene is shown in FIG. 2.
The alphavirus preferably is a Semliki Forest virus and the first DNA sequence is the Semliki Forest viral sequence contained in plasmid pSFV1. The promoter used preferably is the SP6 promoter.
The vector may contain a unique restriction site permitting linearization of the vector without cleaving the second nucleotide sequence and maintaining the first and second nucleotides sequences under transcriptional control of the promoter. The unique restriction site preferably is a SpeI site, particularly that derived from pSFV1. The linearized form of the vector forms an embodiment herein.
The vector may be a plasmid vector, preferably one having the identifying characteristics of plasmid pMP37 (ATCC 97905) as shown in FIG. 1C and, more preferably, is the plasmid pMP37.
The mutant DNA sequence encoding an RSV F protein or a fragment thereof capable of inducing antibodies that specifically react with RSV F protein and lacking the SpeI restriction site present in the native DNA sequence, constitutes another aspect of the present invention. Such mutant DNA sequence lacking the SpeI site of the native sequence preferably is that shown in FIG. 2 (SEQ ID No: 1) or one which encodes the amino acid sequence shown in FIG. 2 (SEQ ID No: 2).
The novel vector provided herein may be linearized by cleavage at the unique restriction site and transcribed to an RNA transcript. In accordance with a further aspect of the invention, there is provided an RNA transcript of a vector as provided herein produced by linearization and transcription.
The RNA transcripts provided herein may be provided in the form of an immunogenic composition for in vivo administration to a host for the generation in the host of antibodies to paramyxovirus protein, such immunogenic compositions comprising, as the active component thereof, an RNA transcript as provided herein. Such immunogenic compositions, which are provided in accordance with another aspect of the invention, may be formulated with any suitable pharmaceutically-acceptable carrier for the in vivo administration and may produce a protective immune response.
In a yet further aspect of the present invention, there is provided a method of immunizing a host against disease caused by infection with paramyxovirus, which comprises administering to the host an effective amount of an RNA transcript as provided herein.
The present invention also includes a novel method of using a gene encoding a RSV F protein or an fragment of an RSV F protein capable of generating antibodies which specifically react with RSV F protein to protect a host against disease caused by infection with respiratory syncytial virus, which comprises isolating said gene; operatively linking said gene to a DNA sequence which is complementary to at least part of an alphavirus RNA genome and having the complement of complete alphavirus RNA genome replication regions in a region of said DNA sequence which is non-essential for replication to form a plasmid vector wherein said gene and DNA sequence are under transcriptional control of a promoter; linearizing the plasmid vector while maintaining said gene and DNA sequence under said transcriptional control of the promoter; forming an RNA transcript of said linearized vector; and introducing said RNA transcript to said host.
Linearizing the plasmid vector is effected by cleaving the plasmid vector at a unique restriction site therein at a location which permits the maintenance of the gene and the DNA sequence under the transcriptional control of the promoter. The unique restriction site may be an SpeI site, such as derived from plasmid pSFV1.
The plasmid vector employed preferably is plasmid pMP37 and the linearizing step is effected by cleavage at the SpeI site of plasmid pMP37 (see FIG. 1C).
In addition, the present invention includes a method of producing a vaccine for protection of a host against disease caused by infection with respiratory syncytial virus (RSV), which comprises isolating a first DNA sequence encoding an RSV F protein from which the transmembrane anchor and cytoplasmic tail are absent and lacking any SpeI restriction site; operatively linking said first DNA sequence to a second DNA sequence which is complementary to at least part of an alphavirus RNA genome and having the complete alphavirus genome replication regions in a region of said second DNA sequence which is non-essential for replication to form a plasmid vector wherein said first and second DNA sequences are under transcriptional control of a promoter; linearizing the plasmid vector while maintaining said first and second DNA sequences under said transcriptional control of the promoter; forming a RNA transcript of said linearized vector; and formulating said RNA transcript as a vaccine for in vivo administration.
Linearizing the plasmid vector is effected by cleaving the plasmid vector at a unique restriction site therein at a location which permits the maintenance of the gene and the DNA sequence under the transcriptional control of the promoter. The unique restriction site may be an SpeI site, such as derived from plasmid pSFV1.
The plasmid vector employed preferably is plasmid pMP37 and the linearizing step is effected by cleavage at the SpeI site of plasmid pMP37.
Advantages of the present invention include the c provision of RNA transcripts which are useful in generating an immune response by in vivo administration.